Vps34 is a lipid kinase that regulates membrane trafficking pathways like endocytosis and autophagy by generating phosphoinositol 3-phosphate, a lipid signaling molecule. Endocytosis is the process by which cells take up extracellular materials and transmembrane proteins, and sort them to their proper destinations. Endocytosis is necessary for cells to acquire nutrients and for appropriate growth control. Autophagy is an evolutionarily conserved process that promotes cell survival by serving a housekeeping role, and by providing extra nutrients when the cell is under stress. As both endocytosis and autophagy are important cellular events in cancer development and resistance to therapy, they have become targets for designing novel cancer therapeutics. Therefore, it is important to understand how these cellular events are regulated. Although Vps34 is implicated in both endocytosis and autophagy, determining its precise role has been hampered by the lack of specific inhibitors. Using a genetic knockout strategy we recently reported that Vps34 has a major role in autophagosome formation and the late stages of endocytosis, but may be dispensable at the early endosome. This proposal is designed to determine 1) the precise role of Vps34 during endocytosis by examining the functions of the early endosome, the process of endosome maturation and the functions of the late endosome in Vps34-deficient cells, and 2) how autophagy is regulated via Vps34 dependent or independent mechanisms. These studies will help provide a clearer picture of the exact roles of Vps34 in endocytosis and autophagy. This information will be critical for understanding the multifaceted functions of Vps34 and for the design of anti-cancer therapeutics targeting endocytosis or autophagy.